Regulation of renal ion channels by serum and glucocorticoid inducible kinase isoforms, ubiquitin ligase Nedd4-2 and NHE3 regulating factor 2 in the Xenopus laevis oocyte expression system [Elektronische Ressource] / eingereicht von Hamdy M. Embark

Regulation of renal ion channels by serum and glucocorticoid inducible kinase isoforms, ubiquitin ligase Nedd4-2 and NHE3 regulating factor 2 in the Xenopus laevis oocyte expression system [Elektronische Ressource] / eingereicht von Hamdy M. Embark

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Regulation of Renal Ion Channels by Serum and Glucocorticoid Inducible Kinase Isoforms, Ubiquitin Ligase Nedd4-2 and NHE3 Regulating Factor 2 in the Xenopus Laevis Oocyte Expression System INAUGURAL-DISSERTATION zur Erlangung des Grades eines Dr. med. vet. beim Fachbereich Veterinärmedizin der Justus-Liebig-Universität Gießen HAMDY M. EMBARK Aus dem Institut für Physiologie der Universität Tübingen Abteilung Physiologie I Betreuer: Prof. Dr. F. Lang Eingereicht über das Institut für Veterinär-Physiologie der Justus-Liebig-Universität Gießen im Fachbereich vertreten durch: Prof. Dr. R. Gerstberger Regulation of Renal Ion Channels by Serum and Glucocorticoid Inducible Kinase Isoforms, Ubiquitin Ligase Nedd4-2 and NHE3 Regulating Factor 2 in the Xenopus Laevis Oocyte Expression System INAUGURAL-DISSERTATION zur Erlangung des Grades eines Dr. med. vet. beim Fachbereich Veterinärmedizin der Justus-Liebig-Universität Gießen Eingereicht von HAMDY M. EMBARK Tierarzt aus Aswan (Ägypten) Gießen 2004 Mit Genehmigung des Fachbereichs Veterinärmedizin der Justus-Liebig-Universität Gießen Dekan: Professor Dr. Dr. h. c. B. Hoffmann Gutachter: Professor Dr. F. Lang Professor Dr. R. Gerstberger Tag der Disputation: 22.

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Regulation of Renal Ion Channels by Serum and Glucocorticoid
Inducible Kinase Isoforms, Ubiquitin Ligase Nedd4-2 and NHE3
Regulating Factor 2 in the Xenopus Laevis Oocyte Expression
System






INAUGURAL-DISSERTATION
zur Erlangung des Grades eines Dr. med. vet.
beim Fachbereich Veterinärmedizin
der Justus-Liebig-Universität Gießen








HAMDY M. EMBARK









Aus dem Institut für Physiologie
der Universität Tübingen
Abteilung Physiologie I
Betreuer: Prof. Dr. F. Lang



Eingereicht über das Institut für Veterinär-Physiologie der
Justus-Liebig-Universität Gießen
im Fachbereich vertreten durch: Prof. Dr. R. Gerstberger



Regulation of Renal Ion Channels by Serum and Glucocorticoid
Inducible Kinase Isoforms, Ubiquitin Ligase Nedd4-2 and NHE3
Regulating Factor 2 in the Xenopus Laevis Oocyte Expression
System



INAUGURAL-DISSERTATION
zur Erlangung des Grades eines Dr. med. vet.
beim Fachbereich Veterinärmedizin
der Justus-Liebig-Universität Gießen



Eingereicht von

HAMDY M. EMBARK
Tierarzt aus
Aswan (Ägypten)



Gießen 2004





Mit Genehmigung des Fachbereichs Veterinärmedizin

der Justus-Liebig-Universität Gießen



































Dekan: Professor Dr. Dr. h. c. B. Hoffmann
Gutachter: Professor Dr. F. Lang
Professor Dr. R. Gerstberger
Tag der Disputation: 22. März 2004





















…to my family


















Contents

List of Abbreviations ....................................................................................................... i
1 INTRODUCTION1
1.1 The renal ion channels ......................................................................................2
+1.2 The renal epithelial K channels ROMK (K 1.1)............2 ir
1.2.1 Physiological roles of ROMK channels in the kidney..............................2
1.2.2 Alternative splicing of the romk (K 1.1 or KCNJ1) gene .........................4 ir
1.2.3 Molecular structure........................................................................................6
1.2.4 Physiological regulation of channel activity...............7
1.2.5 The antenatal Bartter Syndrome (aBS).....................8
2+1.3 The renal epithelial Ca channel ECaC1 (TRPV5)....................................11
1.3.1 Physiological roles of ECaC1 in the kidney............11
1.3.2 Genomic structures of ECAC1 and ECAC2 genes12
1.3.3 Molecular structure......................................................................................13
1.3.4 Physiological regulation of channel activity.............14
1.3.5 Clinical implications of ECaC1 channel regulation................................15
1.4 The renal CIC-K/barttin chloride channels ...................................................16
1.4.1 Expression pattern and physiological functions.....16
1.4.2 Genomic structures of CLCNKA and CLCNKB genes..........................18
1.4.3 Molecular structure......................................................19
1.4.4 .............................................20
1.4.5 Pathophysiological significance of ClC-K/barttin channels ...................21
1.5 Serum and glucocorticoid inducible kinase and protein kinase B ............22
+ +1.6 The Na /H exchanger regulating factor (NHERF).....................................24
1.7 The ubiquitin protein ligase Nedd4 ................................26
1.8 Xenopus laevis oocytes and electrophysiological recording .....................30
1.9 AIMS OF THE PRESENT STUDY................................................................33
2 MATERIALS AND METHODS......34
2.1 Equipment and materials ................................................................................35
2.1.1 Laboratory equipment.................35
2.1.2 Materials .......................................36
2.1.3 Chemicals and reagents............36
2.1.4 Solutions, medium and buffer....................................................................40
2.2 Heterologous expression in Xenopus oocytes............45
2.2.1 In vitro cRNA transcription.........45
2.2.2 Preparation of oocytes................................................................................48
Contents

2.2.3 cRNA injection .............................................................................................48
2.3 Electrophysiological recording.......................................50
2.3.1 Two-electrode voltage-clamp....50
2.3.2 Recording of intracellular pH (pH)............................................................51 i
2.4 Site-directed mutagenesis of ROMK1...........................54
2.5 Deletion of PDZ domains in NHERF2................................57
2.6 Pull-down assays .............................................................57
2.7 Detection of cell surface expression b y chemiluminescence....................58
2.8 Cell surface biotinylation.................................................................................59
2.9 Western blotting and immunohistochemistry for ROMK1 ..........................60
2.10 Immunohistochemistry for CIC-Ka/barttin channels ...................................60
2.11 Uptake measurements ....................................................................................61
2.12 Data evaluation.................................61
3 RESULTS..........................................................................63
+3.1 Regulation of the renal epithelial K channel ROMK1 (K 1.1a)................64 ir
3.1.1 Up-regulation of ROMK1 by SGK1 and NHERF2..................................64
3.1.2 Current-Voltage relationship (I-V) of ROMK1 expressing oocytes......66
3.1.3 Inhibition of ROMK1 by cytosolic acidification........66
3.1.4 pH sensitivity of ROMK1 ............................................................................68
3.1.5 SGK1 determines pH sensitivity of ROMK169
3.1.6 Increase of ROMK1 abundance in the cell membrane .........................74
3.1.7 Influence of SGK1 and NHERF2 coexpression on ROMK1 stability..78
3.1.8 Stimulation of ROMK1 requires second PDZ domain of NHERF2......79
2+3.2 Regulation of the renal epithelial Ca channel ECaC1 (TRPV5) ............85
2+3.2.1 Stimulation of tracer Ca entry via TRPV5.............................................85
2+3.2.2 Inhibitory effect of chelerythrine on tracer Ca entry............................88
2+3.2.3 Inhibition of tracer Ca uptake by ruthenium red..89
2+3.2.4 Ca currents via TRPV5 stimulated by SGK1 and NHERF2 ..............90
3.2.5 TRPV5 activate an endogenous chloride conductance (I )............92 Cl(Ca)
3.2.6 Inhibition of the I by ruthenium red and NPPB................................96 Cl(Ca)
3.2.7 Interaction of TRPV5 and NHERF2 proteins..........98
3.2.8 Stimulation of TRPV5 requires Second PDZ domain of NHERF2 ....100
3.3 Regulation of the renal CIC-Ka/barttin chloride channels........................102
3.3.1 ClC-Ka/barttin induced currents..............................................................102
3.3.2 Regulation of ClC-Ka/barttin channels by Nedd4-2 and SGK1 .........103
3.3.3 -Ka/barttin channels by SGK1 mutants ..................105
Contents

3.3.4 Regulation of ClC-Ka/barttin induced currents by SGK3....................107
3.3.5 Abolished down-regulation of ClCKa/barttin channels by Nedd4-2 ..109
3.3.6 Immunolocalization of ClC-Ka and barttin channels ............................111
4 DISCUSSION..................................................................................................113
+4.1 Regulation of the renal epithelial K channel ROMK1..............................114
+4.1.1 Up-regulation of K transport via ROMK1 by SGK1 and NHERF2 ...114
4.1.2 Determination of pH sensitivity of ROMK1 channel by SGK1............117
4.1.3 Stimulation of ROMK1 requires PDZ domains of NHERF2................119
2+4.2 Regulation of Ca entry via TRPV5 by SGKs and NHERF2..................122
-4.3 Regulation of Cl transport via CIC-Ka/barttin by SGKs and Nedd4-2...125
Summary .....................................................................................................................129
Zusammenfassung ....................................................................................................131
References..................................................................................................................133
Acknowledgments......155
Curriculum Vitae.........................................................................................................157
List of Publications.....158





List of Abbreviations

List of Abbreviations
AMP adenosine monophosphate
ATP adenosine triphosphate
ATPase adenosine triphosphatase
Bq Bequerel
B-RAF protein kinase oncoprotein
BSND Bartter syndrome with sensorineural deafness
cAMP cyclic adenosine monophosphate
?C degree(s) Celsius (centigrade)
cDNA complementary deoxyribonucleic acid
CIC-K kidney chloride channel
cRNA complementary ribonucleic acid
DEPC diethylpyrocarbonate
DMSO dimethylsulfoxide
DNA desoxyribonucleic acid
dNTP desoxyribonucleotidetriphosphate
DTNB 5,5’-dithio-bis[-2-nitrobenzoic acid]
ECaC1 epithelial calcium channel type 1
EDTA ethylene diamine tetra-acetate
EGTA ethyleneglycol-bis (b-aminoethyl)-N, N, N‘, N‘-tetraacetic acid
+ +E equilibrium potential for the ion K K
ELISA enzyme-linked immunoabsorbent assay
+ENaC epithelial Na channel
FBA feedback amplifier
FmocCl 9-fluorenyl-methoxycarbonyl chloride
GST glutathione S-transferase
h human
HEPES N-(2-Hydroxyethyl) piperazine-N-(2-ethanesulfonic acid)
HEK293 human embryonic kidney cell line
HepG2 human hepatoma cell line
IC concentration at which a 50% inhibition is reached 50
IGF-1 insulin-like growth factor 1
I-V current-voltage relation
KCNE1 potassium channel, inwardly rectifying, subfamily E, member 1
KCNJ1 potassium channel, inwardly rectifying, subfamily J, member 1
KCNQ1 rectifying, subfamily Q
kDa kilodalton
K inward-rectifier potassium channels ir
Kv voltage-gated potassium channel
m mouse
M1 membrane-spanning domain 1
M2 -spanning domain 2
mM millimolar
mRNA messenger ribonucleic acid
mV millivolt
µA microampere
9µCi microcurie (1 Ci = 37 x 10 Bq)
List of Abbreviations

µM micromolar
NaPi sodium-dependent phosphate transporter
Nedd4 neuronal precursor cell-expressed developmentally down-
regulated 4
+ +NHE3 Na /H exchanger type 3
+ +NHERF Na /H exchanger type 3 regulating factor
NKCC sodium potassium chloride cotransporter
NMDG N-methyl-D-glucamine
NPPB 5-Nitro-2-(3-phenylpropylamino)benzoic acid
p53 53 kDa tumor suppressor protein
PAGE polyacrylamide gelelectrophorese
PBS phosphate buffer saline
PCR polymerase chain reaction
PD potential difference across the cell membrane
PDZ PSD 95/Drosophila disk large/ZO-1 domain (PDZ domain)
pH intracellular pH i
pk apparent pK app
PKA protein kinase A; cAMP-dependent protein kinase
PKB (Akt) protein kinase B; oncogene from Akt mouse
PKC protein kinase C
pmol picomole
pS picoSiemens
PT proximal tubule
PTH parathyroid hormone
r rat
RNA ribonucleic acid
+ROMK renal outer-medullary K channel
rpm revolutions (rounds) per minute
SDS sodium dodecyl sulfate
SEM standard error of the mean
SGK serum and glucocorticoid inducible kinase
TEA tetraethylammonium
TEVC two-electrode voltage clamp
TRIS Tris(hydroxymethyl)aminomethane
TRPV transient receptor potential (vanilloid family)
U937 human macrophage cell line
V membrane potential m
v/v volume/volume
w/v weight/volume

Introduction




















1 INTRODUCTION

























1